Autologous CD7 CAR-T cells generated without T cell pre-selection in pediatric patients with relapsed/refractory T-ALL: A phase I trial.

Chimeric antigen receptor (CAR)-T cell therapy showed preliminary activity in patients with refractory or relapsed T cell acute lymphoblastic leukemia (r/r T-ALL). However, many obstacles remain, including manufacturing difficulties and risk of infections. This phase I study (NCT04840875) evaluated autologous CD7 CAR-T cells manufactured without pre-selection of healthy T cells in r/r T-ALL. Thirty patients (29 children and one adult) with a median of two lines of prior therapy but without detectable peripheral leukemia were enrolled. Excluding three cases of manufacturing failures, a total of 27 (90%) patients received infusions after products were confirmed free of leukemia contamination, including 16 (59%) meeting planned target doses. Common adverse events within 30 days included grade 3-4 cytopenias (100%), grade 1-2 (70%) and 3-4 (7%, including one dose-limiting toxicity) cytokine release syndrome, grade 1 neurotoxicity (7%), grade 2 infection (4%), and grade 2 graft-versus-host disease (4%). Two patients developed grade 2 infections after day 30. At day 30, 96% responded and 85% achieved complete remission (CR) or CR with incomplete hematologic recovery (CRi). Seventy-four percent underwent transplantation. Twelve-month progression-free survival with and without censoring transplantation was 22% (95% confidence interval 4%-100%) and 57% (41%-81%), respectively. These results support that autologous CD7 CAR-T therapy without T cell pre-selection is feasible in patients with r/r T-ALL.

Identifying risk factors for severe omicron infection in allogeneic hematopoietic stem cell transplant recipients with hematologic malignancies.

BACKGROUND: In December 2022, a large-scale epidemic occurred in China due to Omicron variant of SARS-CoV-2. This study explored risk factors for Omicron infection in transplant recipients at our institution and investigated the factors influencing the severity of SARS-CoV-2 Omicron infection among recipients of allo-HSCT. METHODS: This single-center study investigated totally 63 allogeneic hematopoietic stem cell transplant patients infected with Omicron variant at the Beijing GoBroad Boren Hospital Transplant Center during December 2022 and analyzed their risk factors. RESULTS: The study included 63 allogeneic hematopoietic stem cell transplant patients who developed Omicron infection. There were 34 mild and 29 moderate to severe cases. Their median age was 22 years (range, 1-65 years), with the male-to-female ratio being 1:1.1. Acute myeloid leukemia (53.97%), acute lymphoblastic leukemia (42.86%), and non-Hodgkin lymphoma (3.17%) were underlying diseases. The median time between HCT and Omicron infection was 8.45 months. Significant predictive factors for moderate to severe Omicron infection included older age (p < .0001), cGVHD (p = .0195), concurrent bacterial infection (p < .0001), low absolute lymphocyte count (p = .026), low CD4/CD8 ratio (p = .0091), high CRP (p < .0001), high serum ferritin (p = .0023), high D-dimer (p < .0001), low CD4 absolute count (p = .0057), and low B-cell absolute count (p = .0154). A moderate to high HCT-CI score tended to be associated with moderate to severe infection (p = .0596). CONCLUSION: This study indicates that risk factors for severe Omicron infection include certain clinical characteristics, such as age, cGVHD, and inflammatory response.

C-JUN overexpressing CAR-T cells in acute myeloid leukemia: preclinical characterization and phase I trial.

Chimeric antigen receptor (CAR) T cells show suboptimal efficacy in acute myeloid leukemia (AML). We find that CAR T cells exposed to myeloid leukemia show impaired activation and cytolytic function, accompanied by impaired antigen receptor downstream calcium, ZAP70, ERK, and C-JUN signaling, compared to those exposed to B-cell leukemia. These defects are caused in part by the high expression of CD155 by AML. Overexpressing C-JUN, but not other antigen receptor downstream components, maximally restores anti-tumor function. C-JUN overexpression increases costimulatory molecules and cytokines through reinvigoration of ERK or transcriptional activation, independent of anti-exhaustion. We conduct an open-label, non-randomized, single-arm, phase I trial of C-JUN-overexpressing CAR-T in AML (NCT04835519) with safety and efficacy as primary and secondary endpoints, respectively. Of the four patients treated, one has grade 4 (dose-limiting toxicity) and three have grade 1-2 cytokine release syndrome. Two patients have no detectable bone marrow blasts and one patient has blast reduction after treatment. Thus, overexpressing C-JUN endows CAR-T efficacy in AML.

Impact of ARID1A and TP53 mutations in pediatric refractory or relapsed mature B-Cell lymphoma treated with CAR-T cell therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapy has been used to treat pediatric refractory or relapsed mature B-cell non-Hodgkin lymphoma (r/r MB-NHL) with significantly improved outcomes, but a proportion of patients display no response or experience relapse after treatment. To investigate whether tumor-intrinsic somatic genetic alterations have an impact on CAR-T cell treatment, the genetic features and treatment outcomes of 89 children with MB-NHL were analyzed. METHODS: 89 pediatric patients treated at multiple clinical centers of the China Net Childhood Lymphoma (CNCL) were included in this study. Targeted next-generation sequencing for a panel of lymphoma-related genes was performed on tumor samples. Survival rates and relapse by genetic features and clinical factors were analyzed. Survival curves were calculated using a log-rank (Mantel-Cox) test. The Wilcox sum-rank test and Fisher's exact test were applied to test for group differences. RESULTS: A total of 89 driver genes with somatic mutations were identified. The most frequently mutated genes were TP53 (66%), ID3 (55%), and ARID1A (31%). The incidence of ARID1A mutation and co-mutation of TP53 and ARID1A was high in patients with r/r MB-NHL (P = 0.006; P = 0.018, respectively). CAR-T cell treatment significantly improved survival in r/r MB-NHL patients (P = 0.00081), but patients with ARID1A or ARID1A and TP53 co-mutation had poor survival compared to those without such mutations. CONCLUSION: These results indicate that children with MB-NHL harboring ARID1A or TP53 and ARID1A co-mutation are insensitive to initial conventional chemotherapy and subsequent CAR-T cell treatment. Examination of ARID1A and TP53 mutation status at baseline might have prognostic value, and risk-adapted or more effective therapies should be considered for patients with these high-risk genetic alterations.

Poor outcome of pediatric B-cell acute lymphoblastic leukemia associated with high level of CRLF2 gene expression in distinct molecular subtypes.

Background: Overexpression of the cytokine receptor-like factor 2 (CRLF2) gene is the most common feature in the Philadelphia chromosome (Ph)-like subtype of B-cell acute lymphoblastic leukemia (B-ALL). However, the predictive value of CRLF2 overexpression for the prognosis of pediatric B-ALL patients remain controversial. The molecular mechanisms that upregulate CRLF2 expression level in patients has not been fully elucidated. Methods: In this study, the prognostic impact of CRLF2 expression level on molecular types of B-ALL in pediatric patients from Zhujiang Hospital (n = 111) was retrospectively analyzed. Youden index analysis was used to categorize CRLF2 expression into 3 groups, and these categories more precisely described the differences in the prognosis of patients with varying expression levels of CRLF2 in both the Zhujiang Hospital cohort and the TARGET cohort. Results: We used the Zhujiang Hospital cohort as a discovery cohort to determine the cutoff value of CRLF2 expression. CRLF2-high patients accounted for approximately 6%. In addition, the percentage of bone marrow blast cells and initial white blood cell count in CRLF2-high patients were higher than those in CRLF2-low patients, and MRD turned negative slower. The results were validated in the TARGET cohort and indicated that CRLF2 overexpression could be subdivided by CRLF2 expression levels into 2 categories: CRLF2-high with a poor survival and CRLF2-medium with a good OS and EFS. Such heterogeneity was attributed to the different molecular mechanisms leading to CLRF2 upregulation, where the CRLF2 overexpression level was high in Ph-like B-ALL and medium in high hyperdiploid B-ALL. Conclusion: This study highlights the importance of the molecular mechanisms of the upregulation of CRLF2 expression in predicting the prognosis of pediatric B-ALL patients.

Sequential CD19 and CD22 chimeric antigen receptor T-cell therapy for childhood refractory or relapsed B-cell acute lymphocytic leukaemia: a single-arm, phase 2 study.

BACKGROUND: Relapses frequently occur following CD19-directed chimeric antigen receptor (CAR) T-cell treatment for relapsed or refractory B-cell acute lymphocytic leukaemia in children. We aimed to assess the activity and safety of sequential CD19-directed and CD22-directed CAR T-cell treatments. METHODS: This single-centre, single-arm, phase 2 trial, done at Beijing GoBroad Boren Hospital, Beijing, China, included patients aged 1-18 years who had relapsed or refractory B-cell acute lymphocytic leukaemia with CD19 and CD22 positivity greater than 95% and an Eastern Cooperative Oncology Group performance status of 0-2. Patients were initially infused with CD19-directed CAR T cells intravenously, followed by CD22-directed CAR T-cell infusion after minimal residual disease-negative complete remission (or complete remission with incomplete haematological recovery) was reached and all adverse events (except haematological adverse events) were grade 2 or better. The target dose for each infusion was 0·5 × 106 to 5·0 × 106 cells per kg. The primary endpoint was objective response rate at 3 months after the first infusion. Secondary endpoints were duration of remission, event-free survival, disease-free survival, overall survival, safety, pharmacokinetics, and B-cell quantification. The prespecified activity analysis included patients who received the target dose and the safety analysis included all treated patients. This study is registered with ClinicalTrials.gov, NCT04340154, and enrolment has ended. FINDINGS: Between May 28, 2020, and Aug 16, 2022, 81 participants were enrolled, of whom 31 (38%) were female and 50 (62%) were male. Median age was 8 years (IQR 6-10), all patients were Asian. All 81 patients received the first infusion and 79 (98%) patients received sequential infusions, CD19-directed CAR T cells at a median dose of 2·7 × 106 per kg (IQR 1·1 × 106 to 3·7 × 106) and CD22-directed CAR T cells at a median dose of 2·2 × 106 per kg (1·1 × 106 to 3·7 × 106), with a median interval of 39 days (37-41) between the two infusions. 62 (77%) patients received the target dose, including two patients who did not receive CD22 CAR T cells. At 3 months, 60 (97%, 95% CI 89-100) of the 62 patients who received the target dose had an objective response. Median follow-up was 17·7 months (IQR 11·4-20·9). 18-month event-free survival for patients who received the target dose was 79% (95% CI 66-91), duration of remission was 80% (68-92), and disease-free survival was 80% (68-92) with transplantation censoring; overall survival was 96% (91-100). Common adverse events of grade 3 or 4 between CD19-directed CAR T-cell infusion and 30 days after CD22-directed CAR T-cell infusion included cytopenias (64 [79%] of 81 patients), cytokine release syndrome (15 [19%]), neurotoxicity (four [5%]), and infections (five [6%]). Non-haematological adverse events of grade 3 or worse more than 30 days after CD22-directed CAR T-cell infusion occurred in six (8%) of 79 patients. No treatment-related deaths occurred. CAR T-cell expansion was observed in all patients, with a median peak at 9 days (IQR 7-14) after CD19-directed and 12 days (10-15) after CD22-directed CAR T-cell infusion. At data cutoff, 35 (45%) of 77 evaluable patients had CAR transgenes and 59 (77%) had B-cell aplasia. INTERPRETATION: This sequential strategy induced deep and sustained responses with an acceptable toxicity profile, and thus potentially provides long-term benefits for children with this condition. FUNDING: The National Key Research & Development Program of China, the CAMS Innovation Fund for Medical Sciences (CIFMS), and the Non-Profit Central Research Institute Fund of Chinese Academy of Medical Sciences. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Long-term follow-up of donor-derived CD7 CAR T-cell therapy in patients with T-cell acute lymphoblastic leukemia.

BACKGROUND: Donor-derived CD7-directed chimeric antigen receptor (CAR) T cells showed feasibility and early efficacy in patients with refractory or relapsed T-cell acute lymphoblastic leukemia (r/r T-ALL), in a previous phase I trial report, at a median follow-up of 6.3 months. Here we report long-term safety and activity of the therapy after a 2-year follow-up. METHODS: Participants received CD7-directed CAR T cells derived from prior stem cell transplantation (SCT) donors or from HLA-matched new donors after lymphodepletion. The target dose was 1 × 106 (± 30%) CAR T cells per kg of patient weight. The primary endpoint was safety with efficacy secondary. This report focuses on the long-term follow-up and discusses them in the context of previously reported early outcomes. RESULTS: Twenty participants were enrolled and received infusion with CD7 CAR T cells. After a median follow-up time of 27.0 (range, 24.0-29.3) months, the overall response rate and complete response rate were 95% (19/20 patients) and 85% (17/20 patients), respectively, and 35% (7/20) of patients proceeded to SCT. Six patients experienced disease relapse with a median time-to-relapse of 6 (range, 4.0-10.9) months, and 4 of these 6 patients were found to have lost CD7 expression on tumor cells. Progression-free survival (PFS) and overall survival (OS) rates 24 months after treatment were respectively 36.8% (95% CI, 13.8-59.8%) and 42.3% (95% CI, 18.8-65.8%), with median PFS and OS of respectively 11.0 (95% CI, 6.7-12.5) months and 18.3 (95% CI, 12.5-20.8) months. Previously reported short-term adverse events (< 30 days after treatment) included grade 3-4 cytokine release syndrome (CRS; 10%) and grade 1-2 graft-versus-host disease (GVHD; 60%). Serious adverse events reported > 30 days after treatment included five infections and one grade 4 intestinal GVHD. Despite good CD7 CAR T-cell persistence, non-CAR T and natural killer cells were predominantly CD7-negative and eventually returned to normal levels in about half of the participants. CONCLUSIONS: In this 2-year follow-up analysis, donor-derived CD7 CAR T-cell treatment demonstrated durable efficacy in a subset of patients with r/r T-ALL. Disease relapse was the main cause of treatment failure, and severe infection was a noteworthy late-onset adverse event. TRIAL REGISTRATION: ChiCTR2000034762.

Sequential different B-cell antigen-targeted CAR T-cell therapy for pediatric refractory/relapsed Burkitt lymphoma.

Single antigen-targeted chimeric antigen receptor (CAR) T-cell therapy may be insufficient to induce a durable response in pediatric aggressive B-cell lymphomas. This clinical trial examined the feasibility of sequential different B-cell antigen-targeted CAR T-cell therapy for pediatric relapsed/refractory (R/R) Burkitt lymphoma. Twenty-three patients received the first CD19 CAR T-cell infusion. The patients who did not achieve an ongoing complete response (CR) underwent 1 or more sequential infusions of CAR T-cell therapy that targeted CD22 followed by CD20 according to their disease status and CAR T-cell persistence after each infusion. The median time from the last infusion to the cutoff date was 17 months (range, 15-23 months). The estimated 18-month CR rate was 78% (95% confidence interval [CI], 54%-91%). The estimated 18-month progression-free survival rate was 78% (95% CI, 55%-90%), with 78% (95% CI, 37%-94%) in patients with bulky disease and 60% (95% CI, 25%-83%) in patients with central nervous system (CNS) involvement. During the first CD19 CAR T-cell infusion, grade ≥3 cytokine release syndrome (CRS) occurred in 34.8% and neurotoxicity occurred in 21.7% of all patients. During subsequent infusions, there were only a few incidences of grade >2 CRS and neurotoxicity. All adverse events were reversible. The severity of neurotoxicity was not significantly different between patients with CNS involvement and those who did not have CNS involvement. Sequential CAR T-cell therapy may result in a durable response and is safe in pediatric R/R Burkitt lymphoma. Patients with CNS involvement may benefit from sequential CAR T-cell therapy. This trial was registered at www.chictr.org.cn/index.aspx as #ChiCTR1800014457.

Donor-Derived CD7 Chimeric Antigen Receptor T Cells for T-Cell Acute Lymphoblastic Leukemia: First-in-Human, Phase I Trial.

PURPOSE: Patients with relapsed or refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) have few options and poor prognosis. The aim was to assess donor-derived anti-CD7 chimeric antigen receptor (CAR) T-cell safety and efficacy in patients with r/r T-ALL. METHODS: In this single-center, phase I trial, we administered anti-CD7 CAR T cells, manufactured from either previous stem-cell transplantation donors or new donors, to patients with r/r T-ALL, in single infusions at doses of 5 × 105 or 1 × 106 (±30%) cells per kilogram of body weight. The primary end point was safety with efficacy secondary. RESULTS: Twenty participants received infusions. Adverse events including cytokine release syndrome grade 1-2 occurred in 90% (n = 18) and grade 3-4 in 10% (n = 2), cytopenia grade 3-4 in 100% (n = 20), neurotoxicity grade 1-2 in 15% (n = 3), graft-versus-host disease grade 1-2 in 60% (n = 12), and viral activation grade 1-2 in 20% (n = 4). All adverse events were reversible, except in one patient who died through pulmonary hemorrhage related to fungal pneumonia, which occurred at 5.5 months, postinfusion. Ninety percent (n = 18) achieved complete remission with seven patients proceeding to stem-cell transplantation. At a median follow-up of 6.3 months (range, 4.0-9.2), 15 remained in remission. CAR T cells were still detectable in five of five patients assessed in month 6, postinfusion. Although patients' CD7-positive normal T cells were depleted, CD7-negative T cells expanded and likely alleviated treatment-related T-cell immunodeficiency. CONCLUSION: Among 20 patients with r/r T-ALL enrolled in this trial, donor-derived CD7 CAR T cells exhibited efficient expansion and achieved a high complete remission rate with manageable safety profile. A multicenter, phase II trial of donor-derived CD7 CAR T cells is in progress (NCT04689659).

Diagnosis and treatment of pediatric anaplastic lymphoma kinase-positive large B-cell lymphoma: A case report.

BACKGROUND: Anaplastic lymphoma kinase-positive (ALK+) large B-cell lymphoma (LBCL) is a rare type of lymphoma with high invasiveness and rapid progression. It occurs in all age groups, but is extremely rare in children. The lesions mainly involve the lymph nodes and may present with extra-nodal involvement. Response to conventional chemotherapies and local radiotherapy is poor, with a 5-year overall survival of less than 40%. Recently, the use of ALK inhibitors for the treatment of this disease has been reported. CASE SUMMARY: We present a case of a 12-year-old boy diagnosed with ALK+LBCL. The patient had a 2-mo medical history of a calvarial mass, extensive systemic involvement, and positive bone marrow clathrin heavy chain (CLTC)-ALK fusion gene. Complete remission 1 (CR1) was achieved using the modified LMB89 Group C regimen followed by autologous stem cell transplantation. The patient relapsed 3 mo later. He then achieved CR2 with three short courses of chemotherapy (COP, reduced-dose ICE, low-dose Ara-c+VP16) and continuous alectinib targeted therapy. Afterward, allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed. At 16 mo after the allo-HSCT, the patient was still in CR2. CONCLUSION: The modified LMB89 Group C regimen and ALK inhibitors are effective. Allo-HSCT should be performed after remission.

Complicated pulmonary human coronavirus-NL63 infection after a second allogeneic hematopoietic stem cell transplantation for acute B-lymphocytic leukemia: A case report.

RATIONALE: Viruses are the most common pathogens that can cause infection-related non-recurrent death after transplantation, occurring mostly from the early stages of hematopoietic stem cell transplantation (HSCT) to within 1 year after transplantation. Human coronavirus (HCoV)-NL63 is a coronavirus that could cause mortality among patients with underlying disease complications. Serological tests are of limited diagnostic value in immunocompromised hosts and cases of latent infection reactivation. In contrast, macro-genomic high-throughput (DNA and RNA) sequencing allows for rapid and accurate diagnosis of infecting pathogens for targeted treatment. PATIENT CONCERNS: In this report, we describe a patient who exhibited acute B-lymphocytic leukemia and developed complicated pulmonary HCoV-NL63 infection after a second allogeneic HSCT (allo-HSCT). Six months after the second allo-HSCT, he developed sudden-onset hyperthermia and cough with decreased oxygen saturation. Chest computed tomography (CT) suggested bilateral multiple rounded ground-glass opacities with the pulmonary lobules as units. DIAGNOSES: HCoV-NL63 was detected by metagenomic next-generation sequencing (NGS), and HCoV-NL63 viral pneumonia was diagnosed. INTERVENTIONS: The treatment was mainly based on the use of antiviral therapy, hormone administration, and gamma-globulin. OUTCOMES: After the therapy, the body temperature returned to normal, the chest CT findings had improved on review, and the viral copy number eventually became negative. LESSONS: The latest NGS is an effective method for early infection diagnosis. The HCoV-NL63 virus can cause inflammatory factor storm and alter the neutrophil-to-lymphocyte ratio (NLR). This case suggests that the patient's NLR and cytokine levels could be monitored during the clinical treatment to assess the disease and its treatment outcome in a timely manner.

CD22 CAR T-cell therapy in refractory or relapsed B acute lymphoblastic leukemia.

Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2-99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

Erratum: Preventive effects of kudzu root on bone loss and cartilage degradation in ovariectomized rats.

[This corrects the article on p. 3517 in vol. 9, PMID: 28804568.].

Preventive effects of kudzu root on bone loss and cartilage degradation in ovariectomized rats [corrected].

The clinical utility of Traditional Chinese Medicine (TCM) herbs/roots extracts in osteoporosis (OP) and osteoarthritis (OA) has been described in multiple reports, but there have been few studies of TCM for preventing bone loss and cartilage degradation simultaneously. Six-month-old female Sprague-Dawley rats each were subjected to ovariectomized (OVX) or sham surgery and treated orally once daily with herbal extracts or vehicle. Body weight was recorded weekly, and blood samples were collected from fasting animals at different time points. Biochemical markers of bone resorption and cartilage degradation were analyzed. Changes in bone mineral density and calcium content were determined in the femoral center and femoral telocentric end of rats. Out of 56 TCM herbs/roots extracts, only kudzu root demonstrated consistent joint protective effects. OVX resulted in a marked increase in bone resorption and cartilage degradation, which could be significantly reversed by kudzu after three weeks of treatment. Compared to vehicle, kudzu induced a significant increase in bone mineral density in the femoral center and femoral telocentric end, and calcium content. The results show that kudzu exerts direct effects on articular cartilage in the OVX rat and can effectively prevent the acceleration of cartilage degradation induced by ovariectomy. Moreover, kudzu has demonstrated positive effects on metabolic health (cause a weight reduction) and may represent a possible treatment for OP and OA with high body mass index. Further studies are needed to investigate the potential effects of kudzu root in postmenopausal women.

The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants.

OBJECTIVE: The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors. METHODS: Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF-α) and Oncostatin M (OSM) were cultured for 21 days with or without a number of inhibitors targeting different types of proteases. Monoclonal antibodies were raised against the active sites of ADAMTS-4, -5, MMP-9 and -13, and 4 ELISAs were developed and technically validated. In addition, the established AGNxI (ADAMTS-degraded aggrecan), AGNxII (MMP-degraded aggrecan), and CTX-II (MMP-derived type II collagen) were quantified in the explants-conditioned media. RESULTS: We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-α/ OSM stimulation, whereas no significant active ADAMTS-5 was detected in either extracts or supernatants; ii) Active ADAMTS-4 was primarily responsible for E373-374A bond cleavage in aggrecan in this setting; and iii) The compensatory mechanism could be triggered following the blockage of the enzyme caused by inhibitors. CONCLUSIONS: ADAMTS-4 appeared to be the major protease for the generation of 374ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage breakdown.

Type X collagen levels are elevated in serum from human osteoarthritis patients and associated with biomarkers of cartilage degradation and inflammation.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease, of which the pathogenesis is inadequately understood. Hypertrophy-like changes have been observed as part of the progression of OA. The aim of the study was to develop and characterize a novel biomarker of chondrocytes hypertrophy and investigate how this marker was associated with cartilage degradation and inflammation in patients with various degrees of OA. METHODS: A competitive ELISA, C-Col10, applying a well-characterized monoclonal antibody was developed as a biomarker of chondrocyte hypertrophy through measurement of type X collagen (ColX). The levels of C-Col10, C2M (matrix metalloproteinase-derived fragments of type II collagen) and hsCRP (high sensitive C-reactive protein) were quantified by ELISAs in serum of 271 OA patients stratified by Kellgren-Lawrence (KL) score 0-4. Associations between serum levels of the three biomarkers (log transformed) were analyzed by Pearson's correlation and differences in C-Col10 levels between patients with high and low levels of inflammation measured by hsCRP were analyzed by ANOVA. RESULTS: We developed a C-Col10 assay measuring the C-terminus of ColX. We found significantly higher levels of ColX in patients with KL score 2 compared to patients with no radiographic evidence of OA (KL0) (p = 0.04). Levels of ColX were significantly elevated in OA patients with above normal hsCRP levels (p < 0.0001), as well as significantly correlated with levels of C2M (r = 0.55, p < 0.0001), which suggested that chondrocyte hypertrophy was associated with inflammation and cartilage degradation. There was no correlation between C2M and hsCRP. Age and BMI adjustment didn't change the results. Immuno-staining revealed that ColX was predominately located around the hypertrophic chondrocytes and the clustered chondrocytes indicating that C-Col10 measures may be linked to cartilage hypertrophic changes. CONCLUSIONS: We developed a novel assay, C-Col10, for measurement of chondrocyte hypertrophy and found its levels significantly elevated in OA patients with KL score of 2, and also in OA patients with above normal hsCRP levels. Concentration of C-Col10 strongly correlated with levels of C2M, a marker of cartilage destruction. The data suggest that chondrocyte hypertrophy and subsequent collagen X fragmentation seem to be increased in a subset of patients with inflammatory OA.

The active form of MMP-3 is a marker of synovial inflammation and cartilage turnover in inflammatory joint diseases.

BACKGROUND: Matrix metalloproteinase-3 (MMP-3) plays an important role in the pathology of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Measurement of active MMP-3 in clinical samples could provide information about progression of rheumatoid diseases, and potentially response to treatment. Hence, we aimed to develop a sensitive assay specifically measuring the active form of MMP-3 (act-MMP-3) both in ex vivo models and in human sera. METHODS: A monoclonal antibody against the first 6 amino acids of act-MMP-3 was developed, and the specificity was carefully tested by comparing total and active MMP-3. A technically robust act-MMP-3 ELISA was produced. For biological validation, human synovial membrane and human cartilage explant (HEX) culture models were measured and compared by ELISA and immunoblots. For clinical relevance, the serum levels of act-MMP-3 in AS and RA patients before and after anti-TNF-α treatment were evaluated. RESULTS: A highly specific and technically robust ELISA detecting act-MMP-3 in serum was developed. The lower limit of detection was 33.7 pg/mL. The dilution and spiking recovery of human serum was within 100 ± 20%. The average intra- and inter-assay variations were 3.1% and 13.5% respectively.High levels of act-MMP-3 expression were observed in human synovial membrane culture and oncostatin M and TNF-α stimulated human cartilage. In a cross-sectional study of both AS and RA patients, serum act-MMP-3 level was correlated with C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). In addition, in patients receiving anti-TNF-α treatment, the serum level of act-MMP-3 was significantly reduced compared to baseline level reflecting the anti-inflammatory effects of the treatment. CONCLUSION: We have successfully developed an assay measuring act-MMP-3 in human serum showing correlation to inflammatory markers. Further studies are required to clarify, whether act-MMP-3 can serve as a predictive marker for outcome in chronic rheumatoid disorders.